The overll objective of this project is to determine the molecular and structural mechanisms which regulate microtubule assembly and move chromosomes. Our approach continues to be the development of an isolated mitotic spindle preparation from sea urchin eggs which permits direct analysis of spindle structure, composition, and function. We are also focusing now on the hypothesis that calcium regulates spindle assembly and function in vivo, and that membranes endogenous to the mitotic apparatus locally control intraspindle calcium ion (CA++) concentrations. We have developed a technique which uses an EGTA lysis buffer for isolating and storing membrane-free mitotic spindles that are labile to micromolar CA++. For the EGTA-isolated spindles we intend to continue analyzing the protein composition, ultrastructure, microtubule polarity, and the mechanism by which calcium depolymerizes the microtubules and causes shortening of the spindle fibers. We are investigating optimum buffer conditions for reactivating life-like assembly-disassembly characteristics in the EGTA-isolated spindles and will isolate egg tubulin to use in the reassembly buffers. Tubulin that has been fluorescently labelled with dichlorotriazinyl fluorescein (DTAF) will help us to localize sites of tubulin incorporation and dissociation from the microtubules of EGTA-isolated spindles. We will investigate the incorporation and flux of tubulin in living spindles by microinjecting DTAF-tubulin into mitotic cells. We also intend to isolate mitotic apparatuses that retain the normal structure of the intraspindle membranes, and we will study their ultrastructure and protein composition (comparing them to the membrane-free EGTA-isolated spindles), as well as their ability to sequester CA++. The role of the membranes in regulating intraspindle CA++ concentrations will be investigated in vivo by measuring intracellular CA + fluctuations by CA++ selective microelectrodes, by monitoring changes in chlorotetracycline fluorescence, or aequorin luminescence. Our observations will be made with various microscopy techniques (polarization, darkfield, fluorescence, etc.) and an image-intensified video time-lapse recording system. Ultimately, we hope to achieve an isolated mitotic model system which, with appropriate buffer conditions, will enable us to reactivate life-like chromosome movements.